Development of an in vitro system based on extracts of B.subtilis has begun. This system is capable of the transcription and translation of phage and plasmid DNA. This system will be used in studying gene expression in B. subtilis. Extract preparation and fractionation will be examined to determine the procedures yielding optimum in vitro synthetic activity. The in vitro reaction conditions and the components of the reaction mixture will be varied to determine the optimum synthetic conditions for each template used. The in vitro system will be used to investigate the importance of phage induced modifications to RNA polymerase in regulating the expression of phage gene. It will also be used in studying the expression of plasmid pE194 genes. Most importantly it will be used to study the synthesis of B. subtilis alpha-amylase which is subject to catabolite repression. This will necessitate cloning the alpha-amylase gene which will then serve as the in vitro template. The effect of suspected regulatory molecules on alpha-amylase synthesis will be determined. Potentially with this approach we will be able to elucidate at the molecular level how gene expression is regulated by catabolite repression in B. subtilis.